Designing Coiled Coils for Heterochiral Complexation to Enhance Binding and Enzymatic Stability

Coiled coils, commonly found in native proteins, are helical motifs important for mediating intermolecular interactions. While coiled coils are attractive for use in new therapies and biomaterials, the lack of enzymatic stability of naturally occurring l-peptides may limit their implementation in biological environments. d-peptides are of interest for biomedical applications as they are resistant to enzymatic degradation and recent reports indicate that stereochemistry–driven interactions, achieved by blending d- and l-peptides, yield access to a greater range of binding affinities and a resistance to enzymatic degradation compared to l-peptides alone. To our knowledge, this effect has not been studied in coiled coils. Here, we investigate the effects of blending heterochiral E/K coiled coils, which are a set of coiled coils widely used in biomaterials. We found that we needed to redesign the coiled coils from a repeating pattern of seven amino acids (heptad) to a repeating pattern of 11 amino acids (hendecad) to make them more amenable to heterochiral complex formation. The redesigned hendecad coiled coils form both homochiral and heterochiral complexes, where the heterochiral complexes have stronger heats of binding between the constituent peptides and are more enzymatically stable than the analogous homochiral complexes. Our results highlight the ability to design peptides to make them amenable to heterochiral complexation, so as to achieve desirable properties like increased enzymatic stability and stronger binding. Looking forward, understanding how to engineer peptides to utilize stereochemistry as a materials design tool will be important to the development of next-generation therapeutics and biomaterials.


Peptide Characterization
The coiled coil peptides used in this study were purified by preparative-scale HPLC using the binary gradients listed in Table S1 below.The following figures show chromatograms of crude and purified peptides, as well as MALDI-TOF mass spectra of each purified peptide. .The primary peak corresponding to the purified peptide accounts for >96% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA.  .The primary peak corresponding to the purified peptide accounts for >99% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA.
We note that the MALDI mass spectrum shows a Lys deletion.However, we see only one peak for D-K4 7 in the analytical HPLC chromatogram for the purified peptide.Either the deletion is such a minor product that it is not detected on HPLC (which, despite the size of the peak in the mass spectrum, is possible because MALDI-TOF does not always convey relative abundance), or the deletion is not resolved from the main peptide peak under these HPLC conditions.Regardless, we used unpurified peptides to perform a titration of D-K4 7 into L-E4 7 (Figure S7), which yielded a thermogram and integrated heats of interaction that are very similar to the same titration performed with purified peptides (Figure 2 and Figure S21).Therefore, even if the deletion is present in the purified D-K4 7 , it is unlikely to have affected the conclusions drawn from titrations using this peptide.  .The primary peak corresponding to the purified peptide accounts for >93% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 62% B (v/v) over 17 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA. .The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA.

Figure S12. Analytical HPLC chromatogram of purified L-K3
11 .The primary peak corresponding to the purified peptide accounts for >99% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA. .The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA. .The primary peak corresponding to the purified peptide accounts for >99% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA. .The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA.

Figure S18. Analytical HPLC chromatogram of purified L-E3
11 .The primary peak corresponding to the purified peptide accounts for >97% of the total peak area.The peptide was eluted on a linear AB gradient from 5% to 95% B (v/v) over 9 minutes, where A is ultrapure water + 0.1% TFA and B is acetonitrile + 0.1% TFA.

Determination of CD spectroscopy conditions
The experimental parameters of cuvette path length, buffer concentration, and wavelength range were all varied to find the best experimental conditions for this system.There are three characteristic features of the CD spectrum of an α-helix, including (for an L-peptide), negative peaks at 222 nm and 208 nm as well as a positive peak at 193 nm.Therefore, we initially decided to collect data from 190 nm -250 nm, choosing path length and buffer concentration based on obtaining quality data in this range.To this end, we observed the CD and high tension (HT) voltage signals for data collected in 1 mm and 0.1 mm cuvettes for 1X and 0.1X PBS.HT voltage is used to control the gain of the detector to obtain an ideal signal to noise ratio.When a sample absorbs too much light, the HT increases rapidly, and when HT is ≥ 700, the photons reaching the detector are not sufficient to provide reliable CD data.We found that, when using a path length of 1 mm and 1X PBS, the HT increased rapidly below 200 nm, leading to unreliable CD data, but for 0.1X PBS in a 1 mm cuvette or 1X PBS in a 0.1 mm cuvette, there were no problems with data reliability (Figure S25).Based on this data and in an attempt to keep the buffer concentration consistent with other experiments in this manuscript, we initially decided to use 1X PBS in a 0.1 mm cuvette to take CD measurements.However, we were surprised to find that under these conditions for a 25 μM solution of L-K4 7 , the CD spectrum displayed none of the characteristic peaks for an α-helix (Figure S26, red).For comparison, we also obtained a CD spectrum of 25 μM L-K4 7 in 1X PBS in a 1 mm cuvette and observed the characteristic peaks at 222 nm and 208 nm (Figure S26, black).We also obtained CD spectra of L-K4 7 in 0.1X PBS in both 1 mm and 0.1 mm cuvettes (Figure S26, green and blue) and found that characteristic α-helical peaks were only observed in the 1 mm cuvette.These data indicate that CD signal depends on path length.For this reason, and to keep the buffer concentration consistent with other experiments, we decided to move forward using the 1 mm cuvette and 1X PBS for wavelengths from 200 nm -250 nm, relying on the two characteristic α-helical peaks at 222 nm and 208 nm to demonstrate helicity despite not having reliable data for the characteristic peak at 193 nm.Characteristic peaks for α-helices at 222 nm and 208 nm were only observed in samples measured in a 1 mm cuvette.

CD spectroscopy of blended coiled coils
Upon mixing homochiral or heterochiral blends of heptads and hendecads, we used CD spectroscopy to compare the secondary structure of the blended coiled coils to the individual coiled coils (Figure S28).For homochiral blends, the coiled coils with a heptad repeating sequence were found to have a slightly greater mean residue ellipticity at 208 nm and 222 nm, the wavelengths most associated with α-helicity.The hendecad coiled coils similarly exhibited greater mean residue ellipticities at 208 nm and 222 nm for homochiral blends.On the other hand, heterochiral blends of both heptad and hendecad coiled coils resulted in signals close to zero across the wavelength tested, due to the opposing stereochemistries of the blended peptides.), the complexes remained stable out to 24 or 30 h (Figure S31 and Figure S32)., 3, 6, 12, 30, 48, 72, 120, and 168 h with Proteinase K.

Figure S7 .
Figure S7.Thermogram and integrated binding heat from the titration of unpurified D-K4 7 into L-E4 7 at pH 7.4 in 1X PBS.

Figure S25 .
Figure S25.CD and HT voltage of A) 1X PBS in a 1 mm cuvette, B) 0.1X PBS in a 1 mm cuvette, and C) 1X PBS in a 0.1 mm cuvette.The HT voltage for the 1X PBS in a 1 mm cuvette increases rapidly between 200 nm -190 nm, resulting in unreliable data.The HT voltage remains below 700 V for the other conditions, indicating that the data is reliable for the full range of 190 nm -250 nm.
used to confirm the secondary structure and stereochemistry of the coiled coils used in this study.All coiled coils were helical at 100 μM in 1X PBS (evidenced by peaks at 222 nm and 208 nm), while D-coils exhibit mean residue ellipticities > 0 and L-coils exhibit mean residues ellipticities < 0, consistent with the expected stereochemistry (FigureS27).

Figure S29 .
Figure S29.CD spectra as a function of temperature for A) L-K4 7 : L-E4 7 and B) D-K4 7 : L-E4 7. The top panel shows all CD spectra from 5 °C to 90 °C in intervals of 5 °C and the bottom panel shows just the mean residue ellipticity of the complex at 222 nm as a measure of helicity.

Figure S30 .
Figure S30.CD spectra of A) L-K3 11 : L-E3 11 and B) D-K3 11 : L-E3 11.The top panel shows all CD spectra from 5 °C to 90 °C in intervals of 5 °C and the bottom panel shows just the mean residue ellipticity of the complex at 222 nm as a measure of helicity.
of coiled coils in the absence of Proteinase K As a control experiment, coiled coil complexes were incubated in 1X PBS following the same procedure as the degradation experiment without adding Proteinase K to test their stability in buffer.For heptad complexes (L

Table S1 .
List of elution conditions for preparative-scale HPLC purification of coiled coils.